Computational prediction and experimental validation of microRNAs in the brown alga Ectocarpus siliculosus

We used an in silico approach to predict microRNAs (miRNAs) genome-wide in the brown alga Ectocarpus siliculosus. As brown algae are phylogenetically distant from both animals and land plants, our approach relied on features shared by all known organisms, excluding sequence conservation, genome localization and pattern of base-pairing with the target. We predicted between 500 and 1500 miRNAs candidates, depending on the values of the energetic parameters used to filter the potential precursors. Using quantitative polymerase chain reaction assays, we confirmed the existence of 22 miRNAs among 72 candidates tested, and of 8 predicted precursors. In addition, we compared the expression of miRNAs and their precursors in two life cycle states (sporophyte, gametophyte) and under salt stress. Several miRNA precursors, Argonaute and DICER messenger RNAs were differentially expressed in these conditions. Finally, we analyzed the gene organization and the target functions of the predicted candidates. This showed that E. siliculosus miRNA genes are, like plant miRNA genes, rarely clustered and, like animal miRNA genes, often located in introns. Among the predicted targets, several widely conserved functional domains are significantly overrepresented, like kinesin, nucleotide-binding/APAF-1, R proteins and CED-4 (NB-ARC) and tetratricopeptide repeats. The combination of computational and experimental approaches thus emphasizes the originality of molecular and cellular processes in brown algae.